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Polycyclic aromatic hydrocarbon (PAHs)-degrading bacteria may enhance the bioavailability of PAHs by excreting biosurfactants, by production of extracellular polymeric substances, or by forming biofilms. We tested these hypotheses in pure cultures of PAHs-degrading bacterial strains. Most of the strains did not substantially reduce the surface tension when grown on PAHs in liquid shaken cultures. Thus, pseudo-solubilization of PAHs in biosurfactant micelles seems not to be a general strategy for these isolates to enhance PAHs-bioavailability. Three semi-colloid Sphingomonas polysaccharides all increased the solubility of PAHs (Gellan 1.3- to 5.4-fold, Welan 1.8- to 6.0-fold and Rhamsan 2.4- to 9.0-fold). The increases were most pronounced for the more hydrophobic PAHs. The polysaccharide-sorbed PAHs were bioavailable. Mineralization rates of 9-[14C]-phenanthrene and 3-[14C]-fluoranthene by Sphingobium EPA505, were similar with and without sphingans, indicating that mass-transfer rates from PAHs crystals to the bulk liquid were unaffected by the polysaccharides. Biofilm formation on PAHs crystals may favor the diffusive mass transfer of PAHs from crystals to the bacterial cells. A majority of the PAHs-degraders tested formed biofilms in microtiter wells coated with PAHs crystals. For strains capable of growing on different PAHs; the more soluble the PAHs, the lower the percentage of cells attached. Biofilm formation on PAHs-sources was the predominant mechanism among the tested bacteria to overcome mass transfer limitations when growing on poorly soluble PAHs.

Unstirred boundary layers (UBLs) often act as a bottleneck for the diffusive transport of hydrophobic organic compounds (HOCs) in the environment. Therefore, a microscale technique was developed for quantifying mass transfer through a 100-microm thin UBL, with the medium composition of the UBL as the controllable factor. The model compound fluoranthene had to (1) partition from a contaminated silicone disk (source) into the medium, (2) then diffuse through 100 microm of medium (UBL), and finally (3) partition into a clean silicone layer (sink). The diffusive mass transfer from source to sink was monitored over time by measuring the fluoranthene content of the source and sink disks. The diffusive flux of fluoranthene was slightly higher for air than for water. Cyclodextrin, humic acids, and micelles of sodium dodecyl sulfate (SDS) enhanced the diffusive flux of fluoranthene in water by more than 1 order of magnitude. These results demonstrate that medium constituents, which normally are believed to bind hydrophobic organic chemicals, actually can enhance the diffusive mass transfer of HOCs in the vicinity of a diffusion source (e.g., contaminated soil particles). The technique can be used to evaluate the effect of natural fluids on diffusive mass transfer, as it integrates the different processes, partitioning and diffusion, in one laboratory model.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs approximately 0.1 mg kg(-1)) to heavily polluted industrial sites (total PAHs approximately 400 mg kg(-1)). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of (14)C-labeled spikes. The time to 10% mineralization of added (14)C phenanthrene and (14)C pyrene was inversely correlated with the PAH content of the soils. Substantial (14)C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. (14)C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little (14)C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.

The purpose of this review is to recognize the scientific and environmental importance of diffuse pollution with polycyclic aromatic hydrocarbons (PAHs). Diffuse PAH pollution of surface soil is characterized by large area extents, low PAH concentrations, and the lack of point sources. Urban and pristine topsoils receive a continuous input of pyrogenic PAHs, which induces a microbial potential for PAH degradation. The significance of this potential in relation to black carbon particles, PAH bioaccessibility, microbial PAH degradation, and the fate of diffuse PAHs in soil is discussed. Finally, the state-of-the-art methods for future investigations of the microbial degradation of diffuse PAH pollution are reviewed.

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3-to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.Polycyclic aromatic hydrocarbons (PAHs) are considered ubiquitous pollutants of the environment in industrialized areas. Microbial degradation of PAHs has received much attention as a possible strategy for bioremediation of PAH-contaminated soils. Numerous bacterial strains able to grow on threeand four-ring PAHs have been isolated (3,9,12,18). Assessing the range of different PAHs mineralized by a specific strain is desirable in the characterization of such bacterial isolates. Also, when the degradative community of an environmental sample is described, enumeration of the cells degrading specific PAHs is important.Growth on various PAHs as sole sources of carbon and energy cannot easily be determined with standard growth assays because of the low aqueous solubility and bioavailability of the PAHs, which lead to slow bacterial growth and low cell yields. Traditionally, degradation tests have been done in three ways: (i) by mineralization of radioactive tracers (26), (ii) by formation of clearing zones around colonies growing on agar plates covered with an opaque layer of PAH crystals (12, 15), and (iii) by detection of accumulated colored PAH metabolites (23,27). In this paper we present a new and sensitive microtiter plate method for detection of bacterial growth on crystalline PAHs. The method is based on respiratory reduction of the tetrazolium salt WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate} to a colored formazan by active cells (10). Tetrazolium salts, like WST-1, serve as indicators of dehydrogenase activity as they shift color upon reduction by electrons flowing through the electron transport system and by superoxide radicals produced (20).The principle of our method is as follows. Microtiter plate wel...

Phenanthrene-metabolizing soil microbial communities were characterized by examining mineralization of [ 14 C]phenanthrene, by most-probable-number (MPN) counting, by 16S-23S spacer DNA analysis of the numerically dominant, culturable phenanthrene-degrading isolates, and by examining incorporation of [ 13 C]phenanthrene-derived carbon into sterols and polar lipid fatty acids (PLFAs). An unpolluted agricultural soil, a roadside soil diffusely polluted with polycyclic aromatic hydrocarbons (PAHs), and two highly PAHpolluted soils from industrial sites were analyzed. Microbial phenanthrene degraders were not detected by MPN counting in the agricultural soil and the roadside soil. In the industrial soils, phenanthrene degraders constituted 0.04 and 3.6% of the total number of CFU. 16S-23S spacer DNA analysis followed by partial 16S DNA sequencing of representative isolates from one of the industrial soils showed that one-half of the isolates belonged to the genus Sphingomonas and the other half were closely related to an unclassified beta-proteobacterium. The 13 C-PLFA profiles of the two industrial soils were relatively similar and resembled the profiles of phenanthrene-degrading Sphingomonas reference strains and unclassified beta-proteobacterium isolates but did not match the profiles of Pseudomonas, Mycobacterium, or Nocardia reference strains. The 13 C-PLFA profiles of phenanthrene degraders in the agricultural soil and the roadside soil were different from each other and different from the profiles of the highly polluted industrial soils. Only in the roadside soil were 10me/ 12me18:0 PLFAs enriched in 13 C, suggesting that actinomycetes metabolized phenanthrene in this soil. The 13 C-PLFA profiles of the unpolluted agricultural soil did not resemble the profiles of any of the reference strains. In all of the soils investigated, no excess 13 C was recovered in the 18:26,9 PLFA, suggesting that fungi did not contribute significantly to assimilation of [ 13 C]phenanthrene.

Lectins are a group of diverse proteins which bind to specific configurations of sugar residues. The binding of lectins to sugar residues present in polysaccharides resembles the specific binding of antibodies to antigens (4). Lectins have been widely used to characterize surfaces of eucaryotic cells and polysaccharides. Recently, fluorescently labeled lectins have been applied in the study of biofilm formation and biofilm composition. Excretion of adhesive polymers during attachment of bacterial cells to surfaces has been described using a panel of fluorescent lectins (9,14,20). The formation of biofilms on living and nonliving surfaces has been investigated with lectins (17). Lectins in conjunction with confocal laser scanning microscopy (CLSM) have been valuable tools in the study of the threedimensional structure of biofilms (12,15) or of the composition of extracellular polymeric substances (EPS) involved in accumulation of chlorinated organic compounds (24). Strains of Sphingomonas spp. are known for their interesting catabolic capabilities to degrade a wide variety of environmentally hazardous compounds, including polycyclic aromatics (25), dioxine compounds (6), and chlorinated phenols (3). Theoretically, lectins may be used to study the interaction between Sphingomonas cells and environmental surfaces during biofilm formation or to investigate the interaction of EPS with organic compounds. The common approach has been to deduce the structure or composition of biofilm EPS on the basis of the specific binding of lectins to different sugar residues. In this study, we evaluate the use of lectins for the characterization of Sphingomonas biofilms by investigating the binding of five fluorescent lectins with known specificities to Sphingomonas biofilms and to industrially produced Sphingomonas exopolysaccharides (sphingans) with known molecular structures. MATERIALS AND METHODSBacterial strains and growth conditions. Sphingomonas paucimobilis EPA505 was obtained from J. Mueller (19), and Sphingomonas sp. strain LH128 and Sphingomonas sp. strain LB126 were received from L. Bastiaens (1). All strains were stored in 43% glycerol at Ϫ80°C. The bacteria were grown at room temperature in phosphate minimal medium supplemented with glucose as the sole carbon source (PMMG) containing (in grams/liter) the following: glucose, 2; Na 2 HPO 4 ⅐ 2H 2 O, 0.875; KH 2 PO 4 , 0.1; (NH 4 ) 2 SO 4 , 0.25; MgCl 2 ⅐ 6H 2 O, 0.05; CaCl 2 ⅐ 2H 2 O, 0.015; NaNO 3 , 0.018. The medium was amended with 5 ml of a trace element solution consisting of (in milligrams/liter) the following: Na-EDTA, 800; FeCl 2 , 300; MnCl 2 ⅐ 4H 2 O, 10; CoCl 2 ⅐ 6H 2 O, 4; CuSO 4 , 1; Na 2 MoO 4 ⅐ 2H 2 O, 3; ZnCl 2 , 2; LiCl, 0.5; SnCl 2 ⅐ 2H 2 O, 0.5; H 3 BO 3 , 1; KBr, 2; KI, 2; BaCl 2 , 0.5. Phosphate and glucose were autoclaved separately.Cultivation of biofilms on microscope slides. Single-species biofilms were grown on Cel-Line HTC printed microscope slides with six wells on each slide (Cel-Line Associates, Inc., Newfield, N.J.). The slides were sterili...